AtMYBS1 negatively regulates heat tolerance by directly repressing the expression of MAX1 required for strigolactone biosynthesis in Arabidopsis

Heat stress caused by global warming requires the development of thermotolerant crops to sustain yield. It is necessary to understand the molecular mechanisms that underlie heat tolerance in plants. Strigolactones (SLs) are a class of carotenoid-derived phytohormones that regulate plant development and responses to abiotic or biotic stresses. Although SL biosynthesis and signaling processes are well established, genes that directly regulate SL biosynthesis have rarely been reported. Here, we report that the MYB-like transcription factor AtMYBS1/AtMYBL, whose gene expression is repressed by heat stress, functions as a negative regulator of heat tolerance by directly inhibiting SL biosynthesis in Arabidopsis. Overexpression of AtMYBS1 led to heat hypersensitivity, whereas atmybs1 mutants displayed increased heat tolerance. Expression of MAX1, a critical enzyme in SL biosynthesis, was induced by heat stress and downregulated in AtMYBS1-overexpression (OE) plants but upregulated in atmybs1 mutants. Overexpression of MAX1 in the AtMYBS1-OE background reversed the heat hypersensitivity of AtMYBS1-OE plants. Loss of MAX1 function in the atmyb1 background reversed the heat-tolerant phenotypes of atmyb1 mutants. Yeast one-hybrid assays, chromatin immunoprecipitation‒qPCR, and transgenic analyses demonstrated that AtMYBS1 directly represses MAX1 expression through the MYB binding site in the MAX1 promoter in vivo. The atmybs1d14 double mutant, like d14 mutants, exhibited hypersensitivity to heat stress, indicating the necessary role of SL signaling in AtMYBS1-regulated heat tolerance. Our findings provide new insights into the regulatory network of SL biosynthesis, facilitating the breeding of heat-tolerant crops to improve crop production in a warming world.

MYB proteins are characterized by a highly conserved DNAbinding domain called the MYB domain.This domain generally consists of up to four amino acid sequence repeats (R) of approximately 52 amino acids (Dubos et al., 2010).MYB proteins can be categorized into different subfamilies according to the number of repeats.Plant MYB proteins are divided into four major groups: R2R3-MYB, with two adjacent repeats; R1R2R3-MYB (3R-MYB), with three adjacent repeats; R1R2R2R1/2-MYB (4R-MYB), with four adjacent repeats; and R1/2-MYB, a group of heterogeneous MYB-like (MYBL) proteins that usually but not always contain a single MYB repeat (Dubos et al., 2010).The majority of MYB-family proteins function as transcription factors to affect various aspects of plant growth and responses to biotic and abiotic stresses (Dubos et al., 2010).The AtMYBS1/AtMYBL gene encodes an R1/2-MYB-like protein that was first reported to modulate leaf senescence and the response to abscisic acid (ABA) and salt stress (Zhang et al., 2011).Overexpression of AtMYBS1/AtMYBL enhanced leaf senescence but reduced salt tolerance (Zhang et al., 2011).It was then shown to participate in sugar signaling, similar to its rice homolog OsMYBS1 (Lu et al., 2002;Chen et al., 2017).AtMYBS1/AtMYBL loss-of-function mutants exhibited hypersensitivity to sugars and increased expression of sugarresponsive genes, including genes encoding hexokinase (HXK1), chlorophyll a/b-binding protein (CAB1), and ADP-glucose pyro-phosphorylase (APL3) (Lu et al., 2002).In addition to the sugar pathway, the ABA pathway is also an important pathway regulated by AtMYBS1.Downregulation or loss of function of AtMYBS1 in Arabidopsis results in hypersensitivity to ABA, whereas overexpression of AtMYBS1 causes a reduced response to ABA (Zhang et al., 2011;Chen et al., 2017).
In this study, we demonstrated that AtMYBS1/AtMYBL plays a negative regulatory role in plant heat tolerance by directly inhibiting expression of MAX1, which encodes a critical enzyme in SL biosynthesis.Heat tolerance regulated by AtMYBS1-MAX1 was also found to depend on SL signaling pathways.Our findings provide new insights into the regulatory network of the SL pathway.

AtMYBS1 is a negative regulator of heat tolerance in Arabidopsis
In studies of differentially expressed genes in response to heat stress in Arabidopsis, we found that the MYB-like gene AtMYBS1/AtMYBL (At1g49010) was significantly downregulated during heat treatment (Figure 1A).To confirm the expression pattern of AtMYBS1, we generated transgenic plants harboring the b-glucuronidase (GUS) reporter gene driven by the AtMYBS1 promoter.A GUS activity assay demonstrated a clear reduction in AtMYBS1 expression in response to heat treatment (Supplemental Figure 1A).
To investigate the function of AtMYBS1, we generated AtMYBS1overexpressing (OE) lines (35S:MYBS1) by overexpressing At-MYBS1 driven by the 35S promoter (Supplemental Figure 1B).We also ordered two T-DNA insertion mutants, CS843799 and CS806410, from the SALK mutant collections, which we designated atmybs1-1 and atmybs1-2, respectively (Supplemental Figure 1B).Both of these mutants were null alleles (Supplemental Figure 1B).Phenotypic analyses showed that AtMYBS1-OE plants exhibited hypersensitivity to heat stress compared with wild-type Columbia-0 (Col-0) plants (Figure 1B).By contrast, atmybs1 mutants were more resistant to heat stress (Figure 1B).Consistent with these phenotypic changes, the heat-stress-responsive genes HSF3, HSP70, and HSP90 were downregulated in AtMYBS1-OE plants but upregulated in atmybs1 mutants (Figure 1C).We therefore concluded that AtMYBS1 was a negative regulator of plant heat tolerance.
In addition to their heat-tolerant phenotypes, AtMYBS1-OE plants also had rounder and lighter green leaves (Supplemental Figure 2A), increased branching, and reduced plant height compared with Col-0 plants (Supplemental Figure 2B).The atmybs1 mutants had no significant differences in plant morphology from the Col-0 controls (Supplemental Figure 2A and 2B).Because of similar phenotypes among the different lines, we chose 35S:AtMYBS1#5 and atmybs1#1 to represent AtMYBS1-OE plants and atmybs1 mutants in subsequent studies.

AtMYBS1 negatively regulates MAX1 expression in the regulation of heat tolerance
Based on phenotypic similarities between AtMYBS1-OE plants and SL-related mutants (e.g., dwarf and bushy architecture and rounder and lighter green leaves) (Stirnberg et al., 2002;Waters et al., 2012a), we speculated that overexpression of AtMYBS1 might inhibit the SL pathway.To investigate this hypothesis, we examined the expression of four genes involved in the SL pathway, MAX1 to MAX4, in AtMYBS1-OE plants and atmybs1 mutants.The results showed that MAX1 expression was reduced ($2-fold) in AtMYBS1-OE plants but increased ($2.5fold) in atmybs1 mutants (Figure 2A).MAX2 expression did not differ among these samples (Figure 2A).MAX3 and MAX4 expression was significantly increased ($4-fold) in AtMYBS1-OE plants but slightly decreased in atmybs1 mutants (Figure 2A).Previous studies found that MAX3 and MAX4 were upregulated in max1 and max2 mutants, which might be attributed to negative feedback regulation when SL signaling was suppressed (Bennett et al., 2006;Stirnberg et al., 2007;Brewer et al., 2009;Hayward et al., 2009;Waters et al., 2012b).Based on the above findings, we concluded that AtMYBS1 negatively regulates MAX1 expression in vivo.
To investigate the role of MAX1 in AtMYBS1-regulated heat tolerance, we first examined the pattern of MAX1 expression under heat treatment.The results showed that MAX1 was continuously upregulated during heat treatment (Figure 2B), which was opposite to the AtMYBS1 pattern (Figure 1A and Supplemental Figure 1A).Second, we evaluated the heat tolerance of MAX1 loss-of-function mutants and MAX1-OE transgenic plants (35S:MAX1).The results showed that max1 mutants were sensitive to heat stress, but MAX1-OE plants were tolerant (Figure 2C and Supplemental Figure 3).Third, we overexpressed MAX1 in the AtMYBS1-OE background (35S:MAX1/35S:MYBS1) and found that MAX1 overexpression (35S:MAX1/35S:MYBS1) reversed the heat-sensitive phenotypes of AtMYBS1-OE plants (Figure 2C and Supplemental Figure 4A).In addition to changes in heat tolerance, overexpression of MAX1 in the AtMYBS1-OE background also reversed the dwarf and excessive branching phenotypes of AtMYBS1-OE plants (Supplemental Figure 4B).Finally, we generated atmybs1max1 double mutants and found that loss of function of MAX1 in the atmybs1 background reversed the heat-tolerant phenotypes of atmybs1 mutants (Figure 2C).In summary, we concluded that MAX1 expression was negatively regulated by AtMYBS1 and that MAX1 participated in the regulation of heat tolerance by AtMYBS1.
AtMYBS1 can directly regulate MAX1 through the MYB binding site in the MAX1 promoter To investigate whether MAX1 was the direct target of AtMYBS1, we first performed yeast one-hybrid assays to examine whether AtMYBS1 can bind the MAX1 promoter in vitro.Five truncated MAX1 promoter segments from À2050 to ATG (pMAX1-1 to pMAX1-5) were constructed and examined (Supplemental Figure 5).The results showed that AtMYBS1 could bind the region from À550 bp to À310 bp in the MAX1 promoter (Supplemental Figure 5).Five motifs (MF1 to MF5) were detected in this region (Supplemental Figure 5).To determine which motif interacts with AtMYBS1, we performed assays for nine segments (pMAX1-T1 to pMAX1-T9) with different truncations from À550 bp to À310 bp in the MAX1 promoter.The results (A) Expression patterns of AtMYBS1 in response to heat treatment.Seedlings of wild-type Col-0 grown on half-strength Murashige and Skoog (MS) medium for 14 days were exposed to high temperature in a climate chamber (40 C, 60% humidity, 16 h light/8 h dark cycle) for different times as indicated.At the end of the treatment, plants were collected for RNA extraction, and qRT-PCR was performed to measure AtMYBS1 expression.Three independent biological replicates were performed.Data are means ± SD. (B) Heat tolerances of Col-0 and AtMYBS1-overexpressing plants and atmybs1 mutants.Seedlings of Col-0, AtMYBS1-overexpressing lines (35S:At-MYBS1-2, -5, and -6), and atmybs1 mutant alleles (atmybs1-1 and -2) grown on half-strength MS plates for 14 days in the greenhouse (23 C, 70% humidity, 16 h light/8 h dark cycle) were subjected to heat treatment at 40 C for 6 h in a climate chamber and then recovered at 23 C for 2 h.For survival analysis, plants whose shoot apices turned white were deemed dead.Three biological replicates were performed (n > 50 for each replicate).Data are means ± SD; different letters on error bars indicate significant differences at P < 0.05, Tukey's t-test.(C) Expression of the heat-responsive genes HSF3, Hsp70, and Hsp90 in AtMYBS1-overexpressing lines and atmybs1 mutants.Ten-day-old seedlings of Col-0, AtMYBS1-overexpressing lines (35S:AtMYBS1-2, -5, and -6), and atmybs1 mutants (at-mybs1-1 and -2) grown on half-strength MS plates in a greenhouse (23 C, 70% humidity, 16 h light/8 h dark cycle) were collected for RNA extraction, and qRT-PCR was performed to measure HSF3, HSP70, and HSP90 expression.Three independent biological replicates were performed.Data are means ± SD; different letters on error bars indicate significant differences at P < 0.05, Tukey's t-test.
showed that the MF3 motif between À397 bp and À367 bp was responsible for the interaction (Supplemental Figure 5).Sequence analyses identified an MYB binding site (AACTAAC) in the MF3 motif (Figure 3A and Supplemental Figure 5).To determine whether this MYB binding site was required for the interaction, we deleted it (pMAX1-MD) or introduced an AAC-TAAC to AACTCCG mutation (pMAX1-MP) and found that the interaction disappeared (Figure 3A).This result confirmed the necessary role of the MYB binding site in the interaction between AtMYBS1 and MAX1 in vitro.
To confirm that AtMYBS1 can directly regulate MAX1 in vivo, we performed chromatin immunoprecipitation (ChIP)-qPCR experiments with 35S:AtMYBS1-63HA transgenic plants.The hemagglutinin (HA)-tagged transgenic lines also displayed an increased branching phenotype, indicating that AtMYBS1-63HA functioned normally (Supplemental Figure 6).The ChIP-qPCR results showed that the regions between R4 and R5 encompassing the MYB binding site were significantly enriched (Figure 3B), confirming that AtMYBS1 directly binds the MAX1 promoter through the MYB binding site in vivo.We also performed luciferase (LUC) reporter gene assays in Nicotiana benthamiana to examine the transcriptional repression of MAX1 by AtMYBS1.Two different MAX1 promoters with native (AACTAAC) or mutated (AACTCCG) MYB binding sites were used to drive the expression of luciferase (pMAX1: LUC and pMAX1m:LUC) (Supplemental Figure 7A).The assays showed that LUC signals were significantly repressed by AtMYBS1 when AtMYBS1 and pMAX1:LUC were co-expressed (Figure 3C), but this repression was lost when AtMYBS1 and pMAX1m:LUC were co-expressed (Figure 3C).These results confirmed the transcriptional repression of MAX1 by AtMYBS1 and the role of the MYB binding site in this repression.
To investigate whether the MYB binding site functioned in the regulation of heat tolerance in vivo, we constructed two vectors, pMAX1:gMAX1 and pMAX1m:gMAX1, in which MAX1 promoters with native (AACTAAC) and mutated (AACTCCG) MYB binding sites were used to drive MAX1 expression (Supplemental Figure 7B).We first transformed these two vectors into max1 mutants (pMAX1:gMAX1/max1 and pMAX1m:gMAX1/max1) and evaluated the heat tolerance of the transgenic plants.In pMAX1:gMAX1/max1 plants, MAX1 expression was similar to that in wild-type Col-0 (Supplemental Figure 8), and the transgenic plants displayed no significant difference in heat tolerance from Col-0 (Figure 3D).By contrast, MAX1 expression was significantly enhanced in pMAX1m:gMAX1/max1 plants (Supplemental Figure 8), and the transgenic plants exhibited greater heat tolerance than Col-0 (Figure 3D).(A) MAX1-MAX4 expression levels in Col-0, At-MYBS1-overexpressing plants, and atmybs1 mutants measured by qRT-PCR.Twelve-day-old seedlings of Col-0, the AtMYBS1-overexpressing line 35S:AtMYBS1-5, and the atmybs1 mutant atmybs1-1 grown on half-strength MS plates were collected for RNA extraction, and qRT-PCR was performed to measure MAX1-MAX4 expression.Three independent biological replicates were performed.Data are means ± SD; different letters on error bars indicate significant differences at P < 0.05, Tukey's t-test.(B) MAX1 expression pattern during heat treatment.Ten-day-old seedlings of Col-0 grown in the greenhouse (23 C, 70% humidity, 16 h light/8 h dark cycle) were subjected to heat treatment at 40 C for different times as indicated.qRT-PCR was used to measure MAX1 expression.Three independent biological replicates were performed.Data are means ± SD; different letters on error bars indicate significant differences at P < 0.05, Tukey's t-test.(C) Heat tolerance of MAX1-and AtMYBS1-related plants.Fourteen-day-old seedlings of Col-0, max1-1 mutants, 35S:MAX1#1, 35S:MAX1/35S:AtMYBS1-5#2, 35S:AtMYBS1-5, atmybs1-1, and atmybs1max1-1 double mutants grown in half-strength MS medium in the greenhouse (23 C, 70% humidity, 16 h light/8 h dark cycle) were treated at 40 C for 6 h in a climate chamber and then recovered at 23 C for 2 h in the greenhouse.Plants whose shoot apices turned white were deemed dead.The plant death rates were calculated and statistically analyzed after treatment.Three independent biological replicates were performed (n > 50 plants for each replicate).Data are means ± SD; different letters on error bars indicate significant differences at P < 0.05, Tukey's t-test.
Both pMAX1:gMAX1/max1 and pMAX1m:gMAX1/max1 plants exhibited decreased branching phenotypes compared with max1 mutants, which were similar to those of Col-0 (Supplemental Figure 9).These results confirmed that the MYB binding site in the MAX1 promoter plays a role in the regulation of heat tolerance in vivo.(A) The interaction between AtMYBS1 and the MYB binding site in the MAX1 promoter was detected by yeast one-hybrid assay.Three bait vectors, pAbAi-pMAX1-T10 (the region from À397 to À367 bp in the MAX1 promoter), pAbAi-pMAX1-MD (MYB binding site deleted), and pAbAi-pMAX1-MP (AAC to CCG in the MYB binding site), plus the prey vector pGADT7-AtMYBS1 were cotransformed into yeast strain Y1H Gold and then plated onto specific nutrient-deficient media to test the interactions between AtMYBS1 and the MAX1 promoter.The empty vectors pAbAi and pGADT7 were used as negative controls.The black solid lines indicate sequences incorporated into pAbAi in the MAX1 promoter.The red dotted lines represent deleted sequences.The red letters indicate replaced nucleotides.The numbers indicate the positions of integrated segments corresponding to the MAX1 promoter.
(B) Confirmation of the interaction between AtMYBS1 and the MYB binding site in vivo by chromatin immunoprecipitation (ChIP)-qPCR.ChIP was performed with HA-tagged AtMYBS1-overexpressing plants (35S:AtMYBS1-6XHA) using an HA antibody.Primer pairs (R1f, R1r to R6f, R6r) around the MYB binding site were designed to verify the interaction between AtMYBS1 and the MYB binding site.Accurate locations of primers in the MAX1 promoter are labeled on the right.The inverted triangle represents the AtMYBS1 binding site.Three independent biological replicates were performed.Data are means ± SD; different letters on error bars indicate significant differences at P < 0.05, Tukey's t-test.(C) Transcriptional repression activity of AtMYBS1 determined by luciferase (LUC) reporter gene assays in Nicotiana benthamiana leaf cells.35S:AtMYBS1-GFP, pMAX1:LUC, and pMAX1m:LUC vectors were constructed and co-transformed into N. benthamiana leaf cells.The empty vectors 35S:GFP and LUC (pGreenII0800) were used as internal controls.Three independent biological replicates were performed with similar results.Data are means ± SD; different letters on error bars indicate significant differences at P < 0.05, Tukey's t-test.(D) Functions of MAX1 and MYB binding sites in the regulation of heat tolerance.Fourteen-day-old seedlings of Col-0, max1-1 mutants, pMAX1:gMAX1/max1 transgenic plants (#2, #4, #6), and pMAX1m:gMAX1/max1 transgenic plants (#3, #5, #6) grown in half-strength MS medium in the greenhouse (23 C, 70% humidity, 16 h light/8 h dark cycle) were treated at 40 C for 6 h in a climate chamber, then recovered at 23 C for 2 h in the greenhouse.For survival rate analysis, seedlings whose leaves and shoot apices turned white were deemed dead.Three independent biological replicates were performed (n > 50 for each replicate).Data are means ± SD; different letters on error bars indicate significant differences at P < 0.05, Tukey's t-test.(E) Roles of MAX1 and MYB binding sites in AtMYBS1-regulated plant heat tolerance.Fourteen-day-old seedlings of Col-0, 35S:At-MYBS1max1-1#1, pMAX1:gMAX1/35S:AtMYBS1max1 (#3, #4, #8), and pMAX1m:gMAX1/35S:AtMYBS1max1 (#4, #7, #10) were subjected to heat treatment as described in (C).Death rates were calculated as described in (C).Three independent biological replicates were performed (n > 50 for each replicate).Data are means ± SD; different letters on error bars indicate significant differences at P < 0.05, Tukey's t-test.

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To further examine whether the MYB binding site was responsible for AtMYBS1-regulated heat tolerance in vivo, we transformed the pMAX1:gMAX1 and pMAX1m:gMAX1 vectors into the 35S:At-MYBS1max1 background to generate pMAX1:gMAX1/35S:At-MYBS1max1 and pMAX1m:gMAX1/35S:AtMYBS1max1 transgenic plants.35S:AtMYBS1max1 plants were generated by overexpressing AtMYBS1 under the control of the 35S promoter in the max1 background.In pMAX1:gMAX1/35S:AtMYBS1max1 plants, MAX1 expression was still suppressed (Supplemental Figure 8); the transgenic plants showed heat hypersensitivity and did not differ from 35S:AtMYBS1max1 plants in heat sensitivity (Figure 3E).By contrast, MAX1 expression was significantly enhanced in pMAX1m:gMAX1/35S:AtMYBS1max1 plants (Supplemental Figure 8); the transgenic plants exhibited more heat tolerance than 35S:AtMYBS1max1 plants and even more than Col-0 (Figure 3E).In addition to changes in heat tolerance, pMAX1:gMAX1/35S:AtMYBS1max1 plants still displayed increased branching phenotypes similar to those of AtMYBS1-OE plants (Supplemental Figure 9).By contrast, pMAX1m:gMAX1/35S:AtMYBS1max1 plants exhibited reduced branching similar to that of Col-0 (Supplemental Figure 9).In summary, we concluded that the MYB binding site in the MAX1 promoter was required for regulation of heat tolerance by AtMYBS1-MAX1 in vivo.
AtMYBS1 regulation of heat tolerance depends on SL signaling pathways MAX1 was found to be a critical enzyme in SL biosynthesis (Al-Babili and Bouwmeester, 2015).To investigate whether the AtMYBS1-MAX1 module mediates the regulation of heat tolerance through SL biosynthesis, we first analyzed the role of the SL biosynthesis pathway in regulation of heat tolerance in vivo.In addition to MAX1, we also investigated two other SL biosynthesis genes, MAX3 and MAX4.Gene expression analyses showed that MAX3 and MAX4 exhibited slightly decreased expression in response to heat stress (Supplemental Figure 10).Results of heat treatment showed that, like the max1 mutants, the max3 and max4 mutants were hypersensitive to heat stress (Figure 4A and 4D).Overexpression of MAX3 and MAX4 (35S:MAX3 and 35S:MAX4) conferred heat tolerance (Figure 4A and 4D; Supplemental Figure 11), similar to overexpression of MAX1 (35S:MAX1).We also generated 35S:MAX1/max3 and 35S:MAX1/max4 plants in which MAX1 was overexpressed in the max3 and max4 backgrounds.We found that 35S:MAX1/max3 and 35S:MAX1/max4 plants still exhibited heat hypersensitivity similar to that of the max3 and max4 mutants (Figure 4A and 4D).Application of the SL analog GR24 4DO reversed the heat-hypersensitive phenotypes of the max1, max3, max4, 35S:MAX1/max3, and 35S:MAX1/max4 plants (Figure 4B and 4E).Application of GR24 4DO also reversed the heathypersensitive phenotypes of AtMYBS1-OE and atmybs1max1 plants (Figure 4C and 4F).In addition, MAX1 overexpression in the AtMYBS1-OE background (35S:MAX1/35S:AtMYBS1) reversed the heat-sensitive phenotypes of AtMYBS1-OE plants, and loss of function of MAX1 in the atmybs1 background (atmyb-s1max1 double mutants) reversed the heat-tolerant phenotypes of atmybs1 mutants (Figure 2C).These results indicated that the SL biosynthesis pathway played a positive role in the regulation of heat tolerance and was also required for AtMYBS1-MAX1mediated regulation of heat tolerance.
To determine whether the regulation of heat tolerance by AtMYBS1 occurred through the SL signaling pathway, we first evaluated the heat tolerance of SL receptor d14 mutants (Burger and Chory, 2020; Mashiguchi et al., 2021).The results showed that d14 mutants were hypersensitive to heat stress (Figure 4C and 4F), and GR24 4DO application could not reverse this hypersensitivity (Figure 4C and 4F).Loss of function of D14 in the 35S:MAX1 background (35S:MAX1d14) reversed the heattolerant phenotypes of 35S:MAX1 plants and caused heat hypersensitivity similar to that of d14 mutants (Figure 4C and 4F).These results indicated that SL signaling pathways were involved in the regulation of heat tolerance.Next, to investigate whether SL signaling pathways were involved in AtMYBS1-regulated heat tolerance, we constructed atmybs1d14 double mutants and evaluated their tolerance to heat stress.Heat tolerance of the atmybs1d14 double mutants was significantly lower than that of atmybs1 mutants and similar to that of d14 mutants (Figure 4C and 4F).GR24 4DO application did not reverse the heat-hypersensitive phenotypes of atmybs1d14 mutants (Figure 4C and 4F).In accordance with their different heatresponse behaviors, max1 and atmybs1max1 double mutants showed upregulated expression of the heat-response genes HSF3, HSP70, and HSP90 (Supplemental Figure 12) under GR24 4DO treatment, whereas atmybs1d14 did not show a significant difference (Supplemental Figure 12).In summary, we concluded that the SL signaling pathway was necessary for AtMYBS1-MAX1-mediated regulation of heat tolerance in vivo.

DISCUSSION
SLs are a new class of phytohormones involved in numerous plant physiological processes (Mashiguchi et al., 2021).Impairment of the SL pathway can cause hypersensitivity to several stresses, including drought, salt, and seed thermoinhibition (Mostofa et al., 2018).SL biosynthesis requires D27/AtD27, CCD7 (D17/MAX3/ RMS5/DAD3), CCD8 (D10/MAX4/RMS1/DAD1), and CYP711As (e.g., A1(MAX1)/A2/A3) in a sequential manner (Al-Babili and Bouwmeester, 2015).Among SL biosynthesis enzymes, the CYP711A family, to which MAX1 belongs, plays an essential role in the biosynthesis of both canonical and noncanonical SLs (Mashiguchi et al., 2021).In this study, we revealed that AtMYBS1 functions as a negative regulator of heat tolerance by directly repressing MAX1 expression.Both SL biosynthesis and signaling pathways are required for the regulation of heat tolerance by AtMYBS1.Our results thus provide new information related to SL.
(E) Statistical analysis of survival rates for the plants in (B).After heat treatment, dead plants were counted and statistically analyzed.Plants whose shoot apices turned white were considered dead.Three independent biological replicates were performed (n > 50 for each replicate).Data are means ± SD; different letters on error bars indicate significant differences at P < 0.05, Tukey's t-test.(F) Statistical analysis of survival rates for the plants in (F).After heat treatment, dead plants were counted and statistically analyzed.Plants whose shoot apices turned white were considered dead.Three independent biological replicates were performed (n > 50 for each replicate).Data are means ± SD; different letters on error bars indicate significant differences at P < 0.05, Tukey's t-test.

Plant Communications
The MYB binding site in the MAX1 promoter is responsible for direct repression of MAX1 by AtMYBS1 in regulation of heat tolerance We first found that AtMYBS1 expression was downregulated by heat treatment (Figure 1A and Supplemental Figure 1A), and we then confirmed that AtMYBS1 was a negative regulator of plant heat tolerance (Figure 1B).Phenotypic similarities between SLdeficient/insensitive mutants and AtMYBS1-OE plants prompted us to investigate whether the SL pathway might be regulated by AtMYBS1 in vivo (Brewer et al., 2009;Al-Babili and Bouwmeester, 2015).Our results showed that AtMYBS1 negatively regulates expression of the SL biosynthesis gene MAX1 (Figure 2A).To investigate whether regulation of MAX1 by AtMYBS1 takes part in the regulation of plant heat tolerance, we first examined the role of MAX1 in the regulation of heat tolerance.The MAX1 expression pattern and transgenic studies showed that MAX1 played a positive role in regulating plant heat tolerance (Figure 2B and 2C).To investigate whether AtMYBS1 regulates heat tolerance through MAX1, we evaluated the heat tolerance of 35S:MAX1/ 35S:AtMYBS1 plants and atmybs1max1 double mutants.The results confirmed that AtMYBS1-regulated heat tolerance occurred through negative regulation of MAX1 (Figure 2C).
To determine whether AtMYBS1 can directly regulate MAX1, we investigated their interactions by yeast one-hybrid assays in vitro and ChIP-qPCR in vivo (Figure 3A and 3B; Supplemental Figure 5).The results confirmed the direct interaction between AtMYBS1 and the MAX1 promoter through the MYB binding site.We confirmed the transcriptional repression of MAX1 by AtMYBS1 and the role of the MYB binding site in this repression using LUC reporter gene assays in N. benthamiana leaves (Figure 3C and Supplemental Figure 7A).We then analyzed the role of the MYB binding site in AtMYBS1-MAX1-regulated heat tolerance in two steps.In the first step, we investigated whether the MYB binding site was involved in regulation of heat tolerance by analyzing two types of transgenic plants, pMAX1:gMAX1/max1 and pMAX1m:gMAX1/max1, in which the native promoter and a promoter with a mutated MYB binding site were used to drive MAX1 expression in the max1 mutant background (Supplemental Figure 7B).The results showed that mutation of the MYB binding site interfered with MAX1 repression, confirming the necessary role of the MYB binding site in regulating plant heat tolerance (Figure 3D and Supplemental Figure 8).In the second step, we confirmed the function of the MYB binding site in AtMYBS1regulated heat tolerance.We generated 35S:AtMYBS1max1 plants, in which AtMYBS1 was overexpressed in the max1 background.We then transformed pMAX1:gMAX1 and pMAX1m: gMAX1 vectors into 35S:AtMYBS1max1 plants (pMAX1:gMAX1/ 35S:AtMYBS1max1 and pMAX1m:gMAX1/35S:AtMYBS1max1) and evaluated MAX1 expression and heat tolerance in the transgenic plants.The results showed that mutation of the MYB binding site eliminated AtMYBS1-mediated repression of MAX1, confirming the necessary role of the MYB binding site in AtMYBS1-regulated heat tolerance (Figure 3E and Supplemental Figure 8).

SL biosynthesis and signaling pathways are required for AtMYBS1-regulated heat tolerance
Recent studies have shown that MAX1 is a key enzyme in the SL biosynthesis pathway (Al-Babili and Bouwmeester, 2015).Loss of function of MAX1 leads to impairment of SL biosynthesis and further downstream signaling (Mashiguchi et al., 2021).However, loss of function of an enzyme causes not only a reduction in products but also an accumulation of substrates.Substrate accumulation may also have a large effect on plant development and stress responses.The substrate CL accumulated approximately 700-fold in max1 mutants compared with the control (Al-Babili and Bouwmeester, 2015).Despite having no SL activity, CL has been reported to affect the elongation of plant hypocotyls, indicating that it may have other functions in plants (Scaffidi et al., 2013;Al-Babili and Bouwmeester, 2015).Moreover, the cytochrome P450 enzymes to which MAX1 belongs have been shown to participate in various metabolic processes (Shang and Huang, 2020).Thus, the possibility that MAX1 might be involved in other metabolic pathways in addition to SL biosynthesis cannot be excluded.Based on the above considerations, although AtMYBS1 functions by regulating MAX1 expression, we could not assume that AtMYBS1-regulated heat tolerance must be realized through SL biosynthesis and signaling pathways.We therefore performed further studies to investigate this issue.
In addition to analyzing MAX1, we also analyzed the roles of two other SL biosynthesis genes, MAX3 and MAX4, in the regulation of plant heat tolerance.The MAX3 gene encodes carotenoid cleavage dioxygenase 7 (CCD7), which catalyzes the stereospecific cleavage of 9-cis-b-carotene to produce 9-cis-b-apo-10 0 -carotenal and b-ionone.MAX4 encodes CCD8, which catalyzes the conversion of 9-cis-b-apo-10 0 -carotenal to CL, the substrate of MAX1 (Omoarelojie et al., 2019).We first evaluated the heat tolerance of max3, max4, 35S:MAX3, and 35S:MAX4 plants and found that MAX3 and MAX4 had roles in regulating plant heat tolerance similar to that of MAX1 (Figure 4A).We overexpressed MAX1 in the max3 or max4 background (35S:MAX1/max3 or 35S:MAX1/max3) and found that deficiency in MAX3 and MAX4 products interferes with MAX1 function in regulating heat tolerance (Figure 4A).We also treated max1, max3, max4, 35S:MAX1/max3, 35S:MAX1/max4, AtMYBS1-OE, and atmybs1max1 plants with the SL analog GR24 4DO and found that GR24 4DO reversed the heat hypersensitivity of all these plants (Figure 4B and 4C), confirming the role of SL biosynthesis in regulating plant heat tolerance.These results, combined with those from 35S:MAX1/35S:AtMYBS1 and atmybs1max1 plants (Figure 2C), led us to conclude that the SL biosynthesis pathway was required for AtMYBS1-regulated heat tolerance, although SL contents could not be measured in vivo in Arabidopsis because of technical limitations.
We found that the expression of MAX3 and MAX4 decreased slightly in response to heat stress (Supplemental Figure 10), in contrast to the expression pattern of MAX1 (Figure 2B).However, the degree of change in MAX3 and MAX4 expression was much smaller than that in MAX1 (Figure 2B and Supplemental Figure 10).We speculated that the downregulation of MAX3 and MAX4 might be indirect and due to negative feedback regulation by the upregulation of MAX1 in response to heat stress.The expression of MAX3 and MAX4 was significantly enhanced in AtMYBS1-OE plants but slightly decreased in atmybs1 mutants (Figure 2A).Increased expression levels of MAX3/CCD7 and MAX4/CCD8 were previously reported in SL-deficient and SLinsensitive mutants of several plant species, such as Arabidopsis,

Plant Communications
AtMYBS1 negatively regulates heat tolerance pea, petunia, and rice (Foo et al., 2005;Snowden et al., 2005;Johnson et al., 2006;Umehara et al., 2008;Arite et al., 2009;Drummond et al., 2009;Hayward et al., 2009;Mashiguchi et al., 2009).Upregulation of CCD7 and CCD8 could be reversibly counteracted by exogenous application of GR24, a synthetic SL analog, in wild-type and SL-deficient plants (Umehara et al., 2008;Mashiguchi et al., 2009).Levels of 4DO and SL biosynthetic intermediates such as CL and CLA were also markedly increased in SL-insensitive mutants of Arabidopsis or rice (Umehara et al., 2008;Arite et al., 2009;Abe et al., 2014;Seto et al., 2014).This evidence strongly supports the notion that SL biosynthesis is controlled by negative feedback regulation.Because SLs are involved in the regulation of various plant activities, their levels should be carefully modulated as part of a homeostatic steady state, which might explain the significance of the negative feedback mechanism of SL biosynthesis (Koltai and Beveridge, 2013).Among SL biosynthesis enzymes, the cytochrome P450 enzyme MAX1 and its homologs are essential and convert CL to CLA, which is further processed into diverse canonical and noncanonical SLs (Zhang et al., 2014;Yoneyama et al., 2018;Wakabayashi et al., 2019;Burger, 2021).Thus, the most efficient way to regulate SL biosynthesis might be through direct control of MAX1 expression or MAX1 enzyme activity, which may have significance for plant adaptation to rapidly changing conditions.The actual molecular mechanisms that underlie different expression patterns of MAX3, MAX4, and MAX1 in response to heat stress are interesting and need to be elucidated in future studies.
To investigate whether AtMYBS1-MAX1-regulated heat tolerance depends on the SL signaling pathway, we performed studies on the SL receptor gene d14, which encodes an a/b-hydrolase (Waters et al., 2017).We first wanted to determine the roles of d14 in SL-regulated heat tolerance.Evaluation of heat tolerance in d14, 35S:MAX1d14, and atmybs1d14 plants with or without GR24 4DO treatment revealed that d14 was required for SL-mediated regulation of heat tolerance (Figure 4C and 4F).We next investigated the role of d14 in AtMYBS1-regulated heat tolerance by evaluating the heat tolerance of atmybs1d14 double mutants with or without GR24 4DO application (Figure 4C  and 4F).The results confirmed the necessary role of the SL signaling pathway in AtMYBS1-MAX1-regulated heat tolerance.

Molecular mechanisms underlying the regulation of heat and salt stress responses by AtMYBS1
Previous studies have shown that AtMYBS1/AtMYBL functions as a transcription factor involved in responses to salt stress by regulating the ABA and sugar pathways (Lu et al., 2002;Zhang et al., 2011;Chen et al., 2017).AtMYBS1-OE transgenic plants had an improved seed germination rate under salt-stress conditions (Zhang et al., 2011).However, when the survival rates of 14-day-old seedlings were evaluated, AtMYBS1-OE plants displayed salt-sensitive phenotypes, whereas the atmybs1 mutant was resistant (Zhang et al., 2011).Accordingly, expression of the stress marker genes RD29A and RD29B was decreased in AtMYBS1-OE plants but increased in atmybs1 mutants (Zhang et al., 2011).The different seed germination phenotypes and survival rates of 14-day-old seedlings under salt stress indicated that AtMYBS1 might function developmentally in regulating stress sensitivity.The atmybs1 mutants were hypersensitive to ABA, and the ABA biosynthesis genes ABA1, NECD9, and AAO3 and the ABA signaling genes ABI3, ABI4, and ABI5 were upregulated (Chen et al., 2017).These results indicated that atmybs1 mutants might have an increased level of ABA in vivo (Chen et al., 2017).In addition, expression of HXK1, a glucose sensor, was increased in atmybs1 mutants, indicating that AtMYBS1 might negatively regulate the sugar pathway (Rolland et al., 2006;Chen et al., 2017).Previous studies have shown that glucose enhances the ABA pathway through the HXK-dependent sugar signaling pathway (Arenas-Huertero et al., 2000;Cheng et al., 2002).Therefore, enhancement of the ABA pathway in the atmybs1 mutant might be due to increased expression of HXK1.In our studies, seedling survival rates, but not seed germination rates, were evaluated for their tolerance to heat stress.Our results were similar to those of previous studies in which overexpression of AtMYBS1 resulted in hypersensitivity to stress and loss of function of AtMYBS1 resulted in resistance, confirming the negative role of AtMYBS1 in regulating plant stress responses (Zhang et al., 2011).
AtMYBS1 was downregulated by heat stress in our study, but it was induced by salt stress in previous work (Zhang et al., 2011).Salt stress may cause osmotic stress and ionic toxicity (Munns and Tester, 2008).When osmotic stress occurs, plants close the stomata to reduce transpirational water loss (Munemasa et al., 2015).By contrast, plants open the stomata when heat stress occurs and benefit from increased evaporative cooling (Urban et al., 2017).Therefore, different molecular mechanisms may underlie the responses to these two stresses.This may explain why AtMYBS1 exhibited different expression patterns in response to salt and heat stresses, a possibility that will require further investigation in the future.
ABA is a stress hormone that plays an important role in regulating plant responses to different stresses (Bharath et al., 2021).The SL pathway was also found to interact with the ABA pathway.SL has been reported to induce the expression of HB40, which directly activates transcription of the ABA biosynthesis gene AtNCED3 (Gonzalez-Grandio et al., 2017;Wang et al., 2020).In our studies, we found that AtMYBS1 negatively regulates the SL pathway.Thus, the ABA pathway may have been influenced by AtMYBS1, although this will need to be confirmed in future studies.As a transcription factor, AtMYBS1 may have thousands of target genes.For example, the AtMYBS1 homolog in rice was shown to bind to the promoter of a-amylase in vitro (Lu et al., 2002).Comprehensive analysis of AtMYBS1 target genes by ChIP sequencing may be helpful for elucidating the regulatory network of AtMYBS1 in response to different stresses.The roles of AtMYBS1 in regulating heat tolerance and branch number have not been reported previously, and our results provide new insights into the function of AtMYBS1.
On the basis of our results, we propose a functional model for the regulation of heat tolerance by AtMYBS1 in Arabidopsis (Figure 5).Expression of AtMYBS1 is downregulated by heat stress, releasing the direct repression of MAX1 by AtMYBS1 through the MYB binding site in the MAX1 promoter.Increased MAX1 expression activates heat-resistance mechanisms through the SL signaling pathway and confers heat resistance to plants.

Plant Communications
Our studies thus add to the current understanding of the SL pathway in plant development and stress responses.

Plasmid construction and plant transformation
All constructs in this study were created using the Vazyme one-step cloning kit (Vazyme, China, cat.#C115-01).Primers for plasmid construction are listed in Supplemental Table 1.Plant transformation was performed by the floral dip method (Clough and Bent, 1998).In brief, to generate the pMAX1m:LUC plasmid, primers pMAX1m-pGreenII0800-F1 and pMAX1m-pGreenII0800-R1 were used to amplify the first fragment of the MAX1 promoter (Supplemental Table 1), and primers pMAX1m-pGreenII0800-F2 and pMAX1m-pGreenII0800-R2 were used to amplify the second fragment of the MAX1 promoter (Supplemental Table 1).The two fragments were recovered and mixed as templates to amplify the mutated MAX1 promoter using primers pMAX1m-pGreenII0800-F1 and pMAX1m-pGreenII0800-R2.The amplified mutated MAX1 promoter was then integrated into the pGreenII0800 vector.To generate the pMAX1m:g-MAX1 plasmid, primers pMAX1m-gMAX1-F1 and pMAX1m-gMAX1-R1 were used to amplify the first part of the MAX1 genomic sequence, and primers pMAX1m-gMAX1-F2 and pMAX1m-gMAX1-R2 were used to amplify the second part of the MAX1 genomic sequence (Supplemental Table 1).These two fragments were then recovered and mixed to amplify the mutated MAX1 genomic fragment.Finally, the mutated MAX1 genomic fragment was integrated into the pCAMBIA1300 vector.

Heat treatment, GR24 4DO application, branching phenotype observation, and statistical analysis
To examine gene expression patterns, 12-day-old seedlings grown at 23 C in half-strength MS medium were exposed to 40 C in a climate chamber (40 C, 60% humidity, 80-100 mE m À2 s À1 , 16 h light/8 h dark cycle) for the indicated times, then used for qRT-PCR or GUS staining assays.
For heat-tolerance analysis, heat treatments were performed as described in a previous study, with minor modifications (Hong and Vierling, 2000).In brief, seeds of different genotypes were sterilized, sown onto half-strength MS medium, and grown in the greenhouse (23 C, 75% humidity, 60-80 mE m À2 s À1 , 16 h light/8 h dark cycle).Seedlings with two true leaves (14 days) were exposed to 40 C for 6 h in a

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AtMYBS1 negatively regulates heat tolerance climate chamber (40 C, 60% humidity, 80-100 mE m À2 s À1 , 16 h light/8 h dark cycle), followed by 2 h of recovery at 23 C in the greenhouse.For survival analysis, plants whose shoot apices had turned white were considered to be dead.Plant death rates were calculated and statistically analyzed.The original whole-dish photos for Figures 1B, 2C, 3D, 3E, and 4A-4C are provided in Supplemental Figure 13.
For SL treatment, GR24 4DO was purchased from StrigoLab (Italy, cat.#EN4) and dissolved in isopropanol to prepare 0.1 mM solutions.Fourteen-day-old seedlings of different genotypes grown on halfstrength MS medium in the greenhouse (23 C, 75% humidity, 60-80 mE m À2 s À1 , 16 h light/8 h dark cycle) were sprayed with 10 mM GR24 4DO .The treated plants continued to grow in the greenhouse for 8 h (overnight).Subsequently, the GR24 4DO -treated plants were subjected to heat-stress treatment.
For branching observation and statistical analysis, as described in a previous report (Brewer et al., 2016), buds with lengths over 5 mm were defined as newly developed branches.Seeds were sown and grown in the greenhouse (23 C, 75% humidity, 60-80 mE m À2 s À1 , 16 h light/8 h dark cycle).The different lines did not show large differences in flowering time.After all plants had bolted and flowered (approximately 6 weeks, primary branch over 10 cm and self-fertilized), samples (n > 10) were collected, and their primary branches were counted and statistically analyzed.All statistical analyses were performed using Tukey's t-test.

RNA extraction and qRT-PCR analysis
Total RNA was extracted from 2-week-old seedlings with or without heat treatment using an RNAprep Pure Plant Kit (Tiangen, China, cat.#DP441).cDNA was synthesized according to the manufacturer's instructions (Clontech, Japan, cat.#6110A), and qRT-PCR was performed on a 484 ABI 7500 real-time PCR system using the SYBR Green Mix Kit (Bio-Rad, Hercules, CA, USA).ACTIN7 (At5g09180) was used as an internal control.Primers for qRT-PCR are listed in Supplemental Table 1.

GUS staining and activity assay
For GUS staining, a 2613-bp genomic fragment upstream of ATG at the AtMYBS1 locus was amplified, integrated into the pKGWFS7 vector, and transformed into Col-0.GUS staining was performed as described previously (Li et al., 2020).
For the GUS activity assay, GUS activity was quantified using 4-methylumbelliferyl b-D-glucuronide (4-MUG) as the substrate.First, we collected 500 mg of seedling tissue for each sample and ground it into fine powder in liquid nitrogen; we then added 150 ml of GUS extraction buffer (10 mM EDTA [pH 8.0], 0.1% SDS, 50 mM sodium phosphate [pH 7.0], 0.1% Triton X-100, and 10 mM b-mercaptoethanol, with 25 mg/ml phenylmethylsulfonyl fluoride added before use), centrifuged the samples at 15 000 rpm for 10 min, transferred the supernatants to microtubes, and kept them on ice.Second, we prepared a reaction mix (GUS extraction buffer with 1 mM 4-MUG) for each sample, added 1 ml of reaction buffer to microcentrifuge tubes, and prewarmed the tubes at 37 C; 10 ml of the supernatant was then added to the reaction tubes at 30-s intervals and incubated for 10 min, and 100 ml of reaction solution was added to vials containing 1 M sodium carbonate to stop the reaction.Third, we diluted 4-methyl umbelliferone (4-MU) stock solutions to 100 nM, 200 nM, and 400 nM in order to plot a standard curve at an excitation wavelength of 365 nm, emission wavelength of 455 nm, and filter wavelength of 430 nm.We measured the fluorescence of each sample and calculated the amount of 4-MU according to the standard curve.Finally, we quantified the total protein concentration of each sample and determined the GUS activity.

Yeast one-hybrid assay
A yeast one-hybrid assay was performed according to the manufacturer's instructions (Clontech, Japan, cat.#630491, #630466, #630499).In brief, we first amplified the bait sequences (segments in the MAX1 promoter) and incorporated them into the pAbAi vector.We then used the BstBI restriction enzyme to linearize the constructed vectors and transformed them into the yeast strain Y1H Gold.The bait sequences were integrated into the yeast genome via recombination.After selection on synthetic defined (SD) medium without uracil (SD-Ura), we picked healthy colonies for PCR validation.To avoid false-positive errors, the selected yeast colonies were screened on SD-Ura medium supplemented with an appropriate concentration of aureobasidin A (AbA), in the presence of which yeast cells do not grow.Next, the prey vector pGADT7-AtMYBS1 was generated and transformed into the Y1H Gold strains containing the pAbAi-bait vectors.We used SD-Leu selective medium to select positive colonies and subsequently validated them by PCR amplification.Finally, Y1H Gold yeast strains harboring both the pGADT7-AtMYBS1 and pAbAi-bait vectors were plated on SD-Ura-Leu medium containing 50 ng/ml AbA to examine direct interactions between AtMYBS1 and MAX1 promoters.

Western blotting and ChIP-qPCR
Western blotting and ChIP-qPCR were performed as described previously (An et al., 2017).In brief, for the western blot assay, total proteins were extracted from Col-0 and 35S:AtMYBS1-6XHA#7, separated in a 10% polyacrylamide gel, and transferred onto a polyvinylidene fluoride membrane.After blocking, the membrane was sequentially incubated with primary antibody (anti-HA, Abcam, UK, #ab18181) and secondary antibody (mouse HRP, Abcam, #ab131368) at room temperature for 2 h.The chemiluminescent signal was detected using an Enhanced Chemifluorescent HRP Substrate Kit (Thermo Fisher, USA, cat.#15159).
For the ChIP-qPCR assay, 2 g of 2-week-old seedlings of Col-0 and 35S:At-MYBS1-6XHA#7 were ground to fine powder, crosslinked in 1% formaldehyde for 30 min, and neutralized in 0.125 M glycine.The samples were subjected to cell lysis and shearing by sonication (to reduce the DNA fragments to approximately 500 bp).Prior to co-immunoprecipitation, the samples were cleared with Protein A salmon sperm-coupled agarose (Sigma-Aldrich, USA, cat.#16-157).The chromatin samples were then immunoprecipitated overnight at 4 C with HA antibodies (Abcam, #ab18181).Next, the immunoprecipitated chromatin complexes were incubated with protein A salmon sperm-coupled agarose (Sigma-Aldrich, #16-157) and subjected to a series of washing procedures with low salt concentration buffer, high salt concentration buffer, LiCl buffer, and TE buffer.Finally, the immunoprecipitated chromatin was eluted with elution buffer (1% SDS, 0.1 M NaHCO 3 ).Protein-DNA crosslinking was reversed by incubating the immunoprecipitated complexes at 65 C overnight.DNA was recovered using a QIAquick PCR Purification Kit (Qiagen, USA, cat.#28106) and analyzed by real-time qPCR.ACTIN7 (At5g09810) was used as a nonspecific target gene locus.Primers for qPCR are listed in Supplemental Table 1.

Transcriptional activity assay
Luciferase reporter assays were performed to investigate the transcriptional activity of AtMYBS1.First, the AtMYBS1 coding sequence was cloned and inserted into the PJL12-GFP vector to generate the 35S:At-MYBS1-GFP construct.Second, the MAX1 promoter (2050 bp upstream of ATG) was cloned and inserted into pGreenII0800 to generate the pMAX1:LUC construct.The mutated MYB binding site (AACTCCG) in the MAX1 promoter was also cloned and inserted into pGreenII0800 to generate the pMAX1m:LUC construct.The empty vector PJL12-GFP (35S:GFP) and pGreenII0800 were used as controls.All the above vectors were transformed into Agrobacterium tumefaciens (strain GV310).Before infiltration, the GV3101 strains were harvested and resuspended in 2-(Nmorpholino)ethanesulfonic acid (MES) buffer (10 mM MgCl 2 , 10 mM MES, 20 mM acetosyringone [pH 5.7]) and kept in the dark at room temperature for at least 2 h.For different infiltration sets, equal volumes of strains were mixed and injected into N. benthamiana leaves.The infiltrated leaves were sprayed with 10 mM lucoferin (Promega) at 48 h post infiltration and Plant Communications 4, 100675, November 13 2023 ª 2023 The Authors.

AtMYBS1 negatively regulates heat tolerance
Plant Communications kept in the dark for 5 min before luminescence was recorded using the Nightshade LB 985 in vivo Plant Imaging System (Berthold Technologies, Bad Wildbad, Germany).Three independent biological replicates were examined for each set of assays, and each replicate consisted of four leaves from four separate plants.The LUC reporter assays were repeated three times.

Figure 2 .
Figure 2. MAX1 is negatively regulated by AtMYBS1 during the regulation of heat tolerance.

Figure 3 .
Figure 3. MAX1 is directly targeted by AtMYBS1 through the MYB binding site in the MAX1 promoter.

Figure 5 .
Figure 5. Working model for the regulation of heat tolerance by AtMYBS1-MAX1 in Arabidopsis.AtMYBS1 directly represses MAX1 expression through the MYB binding site in the MAX1 promoter.Heat stress represses AtMYBS1 expression, thereby releasing the repression of MAX1 by AtMYBS1.Increased expression of MAX1 activates the SL pathway and thereafter heat-resistance mechanisms, such as enhanced expression of heat-responsive genes (HSF3, HSP70, HSP9), to confer heat resistance to plants.